The reward pathway links adaptive behavior to the appropriate environmental cues through changes in the release of dopamine. Drugs of abuse bypass the requirement for adaptive behavior by increasing dopamine directly, triggering plasticity that results in addiction. However, the mechanisms that control dopamine release remain poorly understood. Unlike other classical transmitters, dopamine acts as a neuromodulator, and it has remained unclear how it can convey a signal with the temporal resolution required to associate environmental cues with reward. In addition, dopamine undergoes release from dendrite as well as axon, and dendritic dopamine release has been implicated in plasticity of the reward system- The long-term objective of this proposal is thus to understand how the regulation of dopamine release contributes to the role of dopamine neurons in physiology and behavior. The strategy is to characterize the mechanisms that regulate dopamine release: 1) Determine the relationship between dopamine and glutamate release by midbrain dopamine neurons. Dopamine neurons form glutamate synapses in vitro, suggesting a mechanism for rapid, precise, synaptic signaling by these cells. Using VGLUT2 conditional knock-out mice, we have now demonstrated a dual role for glutamate co-release in vivo in dopamine storage and as an independent signal for postsynaptic neurons. We will now use live imaging of cultured midbrain dopamine neurons to characterize further the relationship between release ofthe two transmitters. Since VGLUT2 is highly expressed by ventral tegmental area (VTA) dopamine neurons early in development, we will also determine how the VGLUT2 cKO influences monoamine release and synapse formation in vitro, and extend the analysis in vivo using brain slices. 2) Characterize the properties of dendritic dopamine release. Using a pHluorin-based reporter for the vesicular monoamine transporter VMAT2, we have obsen/ed exocytotic events in the dendrites of cultured midbrain dopamine neurons. We will now use imaging to assess the role of release from dense core vesicles and endosomes, and its regulation by action potentials, calcium and synaptic input We will also examine the signals on VMAT2 responsible for its trafficking. The analysis of glutamate co-release by dopamine neurons will explore novel exocytotic pathways relevant to the role of dopamine neurons in reward, but also to many other neuronal populations which have recently been shown to mediate co-release. Similarly, the analysis of dendritic dopamine release will illuminate a process that may contribute to retrograde signaling and plasticity at many synapses.